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Applied and Environmental Microbiology, October 2000, p. 4266-4271, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Application of 5'-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

Hege Karin Nogva,* Knut Rudi, Kristine Naterstad, Askild Holck, and Dag Lillehaugdagger

MATFORSK, Norwegian Food Research Institute, N-1430 Ås, Norway

Received 13 March 2000/Accepted 27 June 2000

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5'-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenes based on the 5'-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all other Listeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5'-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed.

* Corresponding author. Mailing address: MATFORSK, Osloveien 1, N-1430 Ås, Norway. Phone: 47-64-97-01-00. Fax: 47-64-97-03-33. E-mail: hege.nogva{at}matforsk.no.

dagger Present address: Norwegian Food Control Authority, N-0034 Oslo, Norway.

Applied and Environmental Microbiology, October 2000, p. 4266-4271, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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